1. Field of the Invention
The present invention relates generally to a field of transgenic technology, and more particularly relates to a CD81 and OCLN double transgenic mouse and its construction methods and uses.
2. Description of the Prior Arts
Hepatitis C is widespread in the world, as currently there are about 1.3-1.7 million hepatitis C patients. Nearly 80% HCV infected population develops chronic infections, and some of chronic hepatitis C will progress to liver fibrosis, cirrhosis and liver cancer. The HCV is divided into different hypotypes among different races, and the clinic treatments for various HCV hypotypes are also accordingly different. While no vaccine is currently available, effective prevention and treatment for hepatitis C have become a major health issue in need of solution.
The basic studies of HCV infectious and pathogenic mechanism as well as the development of drugs and vaccines would likely benefit from suitable animal models. Chimpanzees are the only species besides humans that is susceptible to HCV infection. However, small number, high costs, slow reproduction, primate animal welfare and growing ethical concerns will limit access to the chimpanzee model, and thus development of suitable alternatives is critical. So far, the development of small animal model of HCV has made some progress, including:
(1) Full-length transgenic mouse: HCV mouse model is developed by transgening HCV full-length genome or specific protein fragment to mouse genome to construct a transgenic mouse having persistent HCV protein expression (Moriya et al., 1998, The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice, Nat Med 4:1065-1067.). The HCV gene overexpression in the transgenic mouse somatic cell would cause expression pressure on the host cell. Such mouse model only expresses HCV gene fragment and lacks the process of HCV virus particles invasion and replication in the cells, such that its application is very limited.
(2) The tree shrew model: tree shrews are susceptible to HCV infection (Tong et al., 2011 Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infectio, J Virol 85:2793-2802; Xu et al., 2007). However, said infection is a transient infection, and this model is unable to establish a stable and reproducible infection. As the tree shrew is a wild animal, artificial feeding and breeding cannot be easily sustained, and its genetic strains are unstable, making it unsuitable for long-term research and application.
(3) Chimeric mouse model: engrafting primary human liver cells to immunodeficient mice or embedding human liver tissue into renal capsular of the mice can support HCV infection and replication. However, this model is limited by low efficiency of viral infection, human liver tissue or cellular immune rejection, and lack of immune response against the pathology of HCV. For example, urokinase-type plasminogen-activator gene (uPA) expression is regulated by albumin promoter (specifically expressed in liver) in severe combined immunodeficiency (SCID) mice would cause persistent liver damage (Kaul et al., 2007, Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 81:13168-13179).
(4) SCID mice transplanted with human liver cell and immune system has limited HCV expression with partial hepatitis pathological process. Moreover, the technology is complex, SCID mice are not readily available, HCV infection is also unstable, and ethical concerns are involved. For example, FK506 binding protein and caspase 8 fusion protein are regulated by albumin promoter in the immunodeficient Balb/C mice having Rag2−/−IL2rg−/−, such that the mice can induce liver damage and then accept human liver cell transplantation after induction (Washburn et al., 2011, A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease, Gastroenterology 140:1334-1344.).
(5) Studies have shown that the species specification of HCV infection depends on the infected subject. The mouse transplanted with CD81 of HCV cell receptor and OCLN (Occludin) can support virus infection and replication in the cellular level. HCV replication in mouse hepatocytes can be detected by adenovirus vector carrying CD81 and OCLN in mouse hepatocytes after transient expression, but cannot establish infection and hepatitis pathological variation. For example, the four HCV receptor genes carried by adenoviral vector can express in mouse, and induce HCV to be able to enter mouse cell for replication. However, the life-cycle of HCV in this model is not complete and hepatitis pathological processes cannot be duly observed (Dorner et al., 2011, A genetically humanized mouse model for hepatitis C virus infection. Nature 474:208-211.).